ram, on Nov 4 2009, 04:19 AM, said:
i am using a molecular weight marker from Bangalore Genie (India)(Old catalog no. PMWM, new catalog no. 105979). In the image below A is the pattern which the company has given in the catalog, whereas B is the pattern which I got on same- 12% SDS-PAGE. As you can see clearly, there is a difference in the spacing between bands of the 2 patterns? Can anyone comment on why is it so? Is such case can I just count number of the bands and then compare the pattern order wise?
Just plot each protein weigh vs mobility you will probably obtain a curve intead of a straight line, that's ok.
It seems that you got higher resolution of the lower weight proteins this may be due to:
-The inclusion of urea or glicerine in the gel.
-Different buffer in the + and - pole
This is usually an effect of different molarity between the gel buffer and +pole buffer.
By the time the gel is inmersed in the tank, if your gel has lower molarity than the tank buffer it will start to slowly equilibrate by more molecules (Tris probably) entering the gel. This will increase the resistance and thus current on that end of the gel, thus your proteins will run faster on the border of the gel.
If you want to be sure the marker is alright, which it probably is, run BSA or other know weight protein on a parallel lane. Keep the same salt, SDS-detergents, protein qt content on each lane.